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Keywords:

  • CD8+ T cell;
  • immunoglobulin-secreting cell;
  • interferon-gamma;
  • oestrogen;
  • progesterone

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

To understand more clearly how mucosal and systemic immunity is regulated by ovarian steroid hormones during the menstrual cycle, we evaluated the frequency of immunoglobulin- and antibody-secreting cells (ISC, AbSC) in genital tract and systemic lymphoid tissues of normal cycling female rhesus macaques. The frequency of ISC and AbSC was significantly higher in tissues collected from animals in the periovulatory period of the menstrual cycle than in tissues collected from animals at other stages of the cycle. The observed changes were not due to changes in the relative frequency of lymphocyte subsets and B cells in tssues, as these did not change during the menstrual cycle. In vitro, progesterone had a dose-dependent inhibitory effect, and oestrogen had a dose-dependent stimulatory effect on the frequency of ISC in peripheral blood mononuclear cell (PBMC) cultures. The in vitro effect of progesterone and oestrogen on ISC frequency could not be produced by incubating enriched B cells alone with hormone, but required the presence of CD8+ T cells. Following oestrogen stimulation, a CD8+ enriched cell population expressed high levels of IFN-gamma and IL-12. The changes in B cell Ig secretory activity that we document in the tissues of female rhesus macaques during the menstrual cycle is due apparently to the action of ovarian steroid hormones on CD8+ T cells. Thus, CD8+ T cells control B cell secretory activity in both mucosal and systemic immune compartments. Understanding, and eventually manipulating, the CD8+ regulatory cell–B cell interactions in females may produce novel therapeutic approaches for autoimmune diseases and new vaccine strategies to prevent sexually transmitted diseases.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Sex steroid hormones are involved in regulating the immune system [1,2] and autoimmune diseases effect women to a greater degree than men (reviewed in [3]). Multiple sclerosis and rheumatoid arthritis occur between two and three times more often in women and systemic lupus erythematosus affects nine times more women than men (reviewed in [3]). Oestrogen is the defining ovarian steroid hormone and effects of oestrogen are mediated by two distinct intracellular receptors, ER-alpha and ER-beta [4]. It seems that women have a greater susceptibility to autoimmune disease at least in part because they make stronger immune responses. The levels of IgM in a primary response, and IgG in a secondary immune response are higher in sexually competent female mice compared to male mice of equivalent age [5–7]. In humans, plasma IgM levels [8] and peripheral CD4+ T cell counts are higher in women than in men [9].

In addition to stronger immune responses and higher concentrations of antibodies and lymphocytes, regulation of immunity is complex in females because lymphocytes respond to changing concentrations of steroid sex hormones. There is a dramatic reduction in the number of activated and committed B cell precursors in the bone marrow of pregnant mice, and this is thought to be due to the elaboration of sex steroids during pregnancy [10,11]. In mice, long-term exposure to high doses of exogenous oestradiol enhances polyclonal B cell activation [12]. In-vitro, oestrogen enhances non-specific differentiation of human immunoglobulin-secreting cells (ISCs) [13–15] and this oestrogen-mediated enhancement is due to inhibition of suppressor T cells [14]. It should be clear from this very brief review that ovarian steroid hormones can regulate immune cell function.

Ovarian sex steroids levels have a particular influence on female genital tract immunity. In the rat, the stage of the oestrous cycle influences the accumulation of IgA and IgG in uterine secretions [16]. IgA and IgG levels in cervical secretions of healthy women are lowest during the periovulatory stage of the menstrual cycle [17]. Within the narrow window of the periovulatory period, IgA levels in human cervical mucus are maximal 2–3 days before ovulation and drop to their minimum level at ovulation [18]. Thus, mucosal immune responses in the female genital tract, as measured by immunoglobulin (Ig) or antibody levels, vary during the menstrual cycle. We recently demonstrated that the effect of menstrual cycle stage on IgG and IgA levels in cervico-vaginal secretions of macaques is similar to women [19]. Thus, in the cervico-vaginal secretions of both monkeys and women, IgA and IgG levels are highest in the luteal phase and during menstruation and lowest around ovulation. We also demonstrated that the changes in Ig levels are not due to changes in immune cell populations in the genital tract [20]. Thus, it seems most likely that functional differences in B cell activity account for the observed cyclicity of Ig levels in female genital tract secretions.

However, no information is available regarding the role of sex steroids on B cell physiology of primates in vivo. In the present study, we document profound changes in the frequency of ISC and antibody-secreting cells (AbSC) in the mucosal and systemic lymphoid tissues of rhesus monkeys at different stages of the menstrual cycle. Further, progesterone suppresses ISC frequency in monkey PBMC in vitro and oestrogen enhances ISC frequency in vitro. The effect of progesterone and oestrogen on the frequency of ISC in vitro could not be elicited by hormone treatment of enriched-B cells, but required the presence of CD8+ T cells in the cultures. The indirect effect of ovarian steroids on B cell function in vitro is consistent with the observed, menstrual cycle-related, variations in the frequency of ISC and AbSC in lymphoid tissues of female rhesus macaques. The results of these studies provide the first clear link between the effects of ovarian hormones on B cell function in vitro and the relative number of antibody-secreting B cells isolated from tissues at different stages of the menstrual cycle

MATERIALS AND METHODS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Animals

Eighteen captive-bred, parous, cycling female rhesus macaques (Macaca mulatta) from the California Regional Primate Research Center (Table 1) were used in this study. All animals were housed in accordance with the American Association for the Accreditation of Laboratory Animal Care (AAALAC) standards. When necessary, the animals were immobilized with 10 mg/kg of Ketamine HCl (Parke-Davis, Morris Plains, NJ, USA) injected intramuscularly.

Table 1.  Group assignment of macaques according to the day of the menstrual cycle at necropsy examination
      
GroupnAnimal no.Day of menstrual cycle at necropsyAge (years. months)No. of pregnancies
Group I722966111·06
  233121 9·55
  232203 9·86
  238803 8·73
  238594 9·63
  21825512·96
  21967711·95
Group II42325411 9·85
  2575412 5·01
  2332015 9·54
  2367215 9·04
Group III7243652014·05
  2426721 7·92
  2410024 8·03
  2439624 8·82
  2370325 9·94
  2385527 9·83
  237782810·04

Assignment of animals to groups for analysis of ISC and AbSC

The stage of the menstrual cycle at the time of necropsy examination was determined for each animal by assessing the level of hormones in urine and the history of menstrual bleeding. Each animal was assigned to one of three groups (Table 1). Animals that were necropsied between days 1 and 7 of the menstrual cycle (oestrogen and progesterone relatively low; menses and early follicular stage) were assigned to Group I (n = 7). Animals that were necropsied between days 11 and 15 of the menstrual cycle (oestrogen high and progesterone relatively low; periovualtory stage) were assigned to Group II (n = 4). Animals that were necropsied between days 20 and 28 of the menstrual cycle (oestrogen moderate and progesterone high; luteal stage) were assigned to Group III (n = 7). A complete list of animals and assignments is shown in Table 1.

Immunogens, immunization and antibody responses

In order to evaluate anamnestic antigen-specific B cell responses, the monkeys were immunized on days 0, 33 and 7 days before necropsy (approximately days 66–130). Thus, while the interval between the second and third immunizations varied by 9 weeks, the timing between the third immunization and necropsy was constant. Each intramuscular injection (quadriceps muscle) contained 560 μg of purified tetanus toxoid (TT) (Connaught Laboratories, INC. Willowdale, Ontario, Canada) and 1000 μg of keyhole limpet haemocyanin (KLH) (Pierce Inc., Rockford, IL, USA). Each oral immunization contained 100 μg of cholera toxin (CT) (List Biological Laboratories, Inc., Campbell, CA, USA). No adjuvant was added to any of the antigens used for immunization. It is important to note that this immunization series was the first exposure of these animals to KLH and CT. In contrast, as a colony management procedure, all the animals had been repeatedly immunized with TT beginning soon after birth. All of the animals had significant serum antibody titres to TT, KLH and CT 14 days after the second immunization. TT, KLH and CT specific IgG or IgA antibody was also present in the CVS of some animals and the titre of these antibodies was the highest during menstruation and the lowest around ovulation (see [16]). At the time of necropsy, serum anti-TT IgG end-point titres ranged from 1·6 × 105 to 2·56 × 106; serum anti-TT IgA end-point titres ranged from 103 to 104; serum anti-CT IgG end-point titres ranged from 2 × 102 to 1·28 × 105; and 13 of 18 sera had anti-CT IgA end-point titres ranging from 15 to 480. Five of 18 animals had undetectable serum IgA anti-CT antibody at the time of necropsy.

Measurement of urine progesterone and oestradiol levels

Urine samples for hormone analysis were collected and pro-cessed as previously described [19]. In order to determine ovarian hormone levels, daily urinary oestrone conjugates (E1C) and pregnanediol-3-glucuronide (Hygeia [Hy]-PdG) of all monkeys were measured by enzyme immunoassay (EIA) as described previously [19]. To compensate for variations in individual urine sample concentrations, urine samples were normalized by assessing creatinine (Cr) concentration by Taussky’s method [21] as described previously [19].

Isolation of lymphocytes from the genital mucosa

At necropsy, fresh cervix and vagina were collected in complete RPMI medium containing 10% fetal calf serum, 2 mML- glutamine, 100 U/ml penicillin, 0·1 mg/ml streptomycin (Sigma Inc., St Louis, MO, USA) and 1% amphotericin-B (Sigma). Genital mucosal tissues were cut into small pieces (maximum size of 10 × 10 × 5 mm), and placed in 1·2 U/ml Dispase II (Boehringer Mannheim, GmbH, Germany) in complete RPMI medium (3 ml per piece). The tissue–dispase II mixture was incubated in a water bath shaker (New Brunswick Scientific, Inc., Edison, NJ, USA) for 2 h at 37°C and 200 r.p.m. After rinsing, the epithelial layer was removed from the underlying tissue by using forceps and scalpel. The subepithelial tissues were cut into 2 × 5 mm slices (approximately 2 mm thick) and incubated in cell release medium. Cell release medium is complete RPMI 1640 containing 25 mM hepes (Sigma), 5 × 10–3Mβ-mercaptoethanol (Sigma), 0·5 mg/ml collagenase (Sigma), 0·1 mg/ml DNAase (Sigma) and 0·02 mg/ml ciprofloxacin-HCl. The tissue was incubated overnight in a shaking water bath at 37°C and 100 r.p.m. To obtain cell suspensions from the lamina propria, 40 ml of the submucosal tissue suspension were shaken vigorously in a tube for 90 s. The resulting cell suspension was then passed through a 100-mesh sterile stainless steel sieve to remove larger pieces of debris and centrifuged 530 g for 10 min. Subsequently, mononuclear cells were isolated on a discontinuous Percoll (Pharmacia) gradient. Cells were resuspended in 40% Percoll in RPMI medium, layered onto 75% Percoll in PBS, and centrifuged at 530 g for 30 min using the no brake setting.

Isolation of lymphocytes from systemic tissues

Tissues collected at necropsy included multiple LNs (iliac, inguinal, axillary and mesenteric LNs), PBMC, spleen, bone marrow and tonsils. The lymphoid tissues were removed and placed directly into sterile culture medium containing 2 mML-glutamine, 100 U penicillin and 100 μg/ml streptomycin (Sigma-Aldrich) at 4°C. Fresh LNs, spleen and tonsil were cut into 2 × 2 mm pieces (<2 mm thick), and squeezed gently using sterile forceps and tongue depressors. Mononuclear cells from spleen cell suspensions, bone marrow and PBMC were isolated by differential gradient centrifugation using ACCU-paque medium (Accurate Chemical & Scientific Corporation, Westbury, NY, USA).

Enumeration of ISC by ELISPOT assay

To detect IgG-ISC, 96-well nitrocellulose membrane plates (Millipore Corporation, Bedford, MA, USA) were coated with goat antimonkey Ig (Fab + Fc)/7 s (Nordic Laboratories Inc., Capistrano, CA, USA) at a concentration of 10 μg/ml in PBS (100 μl/well). To detect IgA-ISC, the plates were coated with rabbit antimonkey IgA (Nordic Laboratories Inc., Capistrano, CA, USA) diluted 1/1000 in PBS (100 μl/well). The plates were then incubated overnight at 4°C in a humid chamber. RPMI 1640 with 10% fetal calf serum was used to block any nitrocellulose not precoated with the antibodies. Serially diluted single-cell suspensions of the various lymph nodes and tissues were incubated at 37°C, 5% CO2 for 16 h. The cells were removed by vigorous washing and the number of Ig-ISC was determined by developing the plate either with a biotinylated goat anti-monkey IgG(Fc) (Nordic Laboratories Inc.) diluted 1/4000 in 1% BSA-PBS or a biotinylated goat anti-monkey IgA(Fc) (Nordic Laboratories Inc.) diluted 1/1000 in 1% BSA-PBS. This was followed by avidin D-peroxidase (Vector Laboratories, Inc., Burlingame, CA, USA) diluted 1/1000 in 1% FCS-PBS and then goat anti-avidin D- peroxidase (Vector Laboratories Inc.) diluted 1/1000 in 1% FCS-PBS. The plates were developed with a peroxidase substrate containing 3-amino-9-ethylcarboazole (Sigma) and 30% H2O2 (Sigma) in acetate buffer pH 5·0. Spots representing individual antibody-secreting cells were counted using a stereomicroscope (Carl Zeiss, Inc, Thornwood, NY, USA) under 20–40 × magnification; spots that had a fuzzy border and deep red centre were scored positive. The results are expressed as the number of ISC per 106 mononuclear cells. For the in vitro B cell enrichment and CD 8+ T cells depletion studies, the results are expressed as the relative frequency of ISC per 105 CD20+ cells. The numbers reported are the means of duplicate wells. Negative controls in every plate consisted of the cells added to duplicate wells that had not been coated with the anti-monkey IgG or IgA antibodies. Additional controls included the treatment of the cells with cycloheximide (3·6 × 10–4M or 2 × 10–3M, Sigma) during the incubation period. Cycloheximide treatment consistently inhibited ISC and ASC spot formation by more than 95%.

Enumeration of AbSC by ELISPOT assay

Ninety-six-well nitrocellulose membrane plates (Millipore, Corporation, Bedford, MA, USA) were coated with TT (40 limit of flocculation units/ml), CT (4 μg/ml) and KLH (5 μg/ml) diluted in PBS (100 μl/well). The plates were placed at 4°C in a humid chamber overnight. RPMI 1640–10% fetal calf serum was used to block any nitrocellulose not precoated with antigen. Serially diluted single cell suspensions of various lymph nodes and tissues were added and the plates were incubated at 37°C for 16 h. The frequency of AbSC was determined by developing the plate either with biotinylated goat anti-monkey IgA (Fc) diluted 1/1000 in 1% BSA-PBS or biotinylated goat anti-monkey IgG (Fc) diluted 1/4000 in 1% BSA-PBS (Nordic, Laboratories, San Juan Capistrano, CA, USA). The remaining steps were the same as for ISC enumeration, as described above. Results are expressed as the mean number of AbSC per 106 mononuclear cells.

In vitro studies on the effect of progesterone and β-oestradiol on ISC frequency in PBMC

PBMC were isolated from normal male rhesus macaques, counted and resuspended in complete RPMI 1640 medium containing either 10, 100, 1000 ng/ml water soluble progesterone (Sigma) or 10, 100, 100 pg/ml water soluble β-oestradiol (Sigma) or control RPMI 1640. Three fivefold serial dilutions of PBMC starting at 106 PBMC/ml were added to wells coated with anti-monkey IgG and IgA antibody and cultured for 18 h. ISC were detected by the ELISPOT assay as described above. In a separate assay, PBMC were cultured in 24 well culture plate for 24 or 48 h. The PBMC were again counted, centrifuged and resuspended in fresh complete medium containing the same concentrations of progesterone or β-oestradiol as described above. Subsequently, the PBMC were incubated in ELISPOT plates for an additional 18 h.

To determine if steroids act directly on B cells, a B cell isolation kit (MACS, Miltenyi Biotec, Inc. Auburn, CA, USA), was used to remove non-B cells from PBMC with magnetic beads and MS+ columns (Miltenyi Biotec) according to the manufacturer’s instructions. The kit contains beads bound to anti-CD2, CD4, CD11b, CD16, CD36 and anti-IgE antibodies. Our primary goal was to remove all the T cells from the cell suspensions. After the first cycle of depletion more than 97% of CD4+ T cells were eliminated. However, the cell suspensions still contained 8–10% of the original CD8+ T cell population. CD8+ T cells were depleted further using anti-CD8+ T cell magnetic beads (Dynal Inc., NY, USA) at a bead to cell ratio of 8 : 1. Following this procedure, T cells comprised less than 3% of the cells in the B cell-enriched PBMC fraction.

To produce CD8+ T cell depleted-PBMC for use in subsequent ELISPOT e×periments, CD8+ T cells were removed specifically from PBMC using two rounds of anti-CD8 magnetic bead (Dynal Inc.) selection. Less than 2% of the cells remaining after this procedure were CD8+ (data not shown). The B cell enriched-PBMC and CD8+ T cell depleted-PBMC were counted, treated with various concentrations of progesterone or estradiol, and immediately applied to ELISPOT assay as described above.

Because antibody binding can dramatically effect gene expression in lymphocytes, a negative selection technique with magnetic beads was used to produce the CD8 negative cell fraction and the CD8 positive cell fraction of hormone-treated PBMC used to assess gene expression following hormone stimulation. CD8+ and CD8 cell fractions were isolated using the human CD8 T Cell Isolation Kit by Miltenyi (Miltenyi) according to the manufacturer’s protocol. The kit contains beads bound to anti-CD4, CD11b, CD16, CD19, CD36 and CD56 antibodies. Less than 10% of the cells in the CD8+ T cell depleted population were CD8+ (data not shown). The CD8+ T cell-enriched cell population contained appro×imately 80% CD3+CD8+ cells, 5% CD3CD8+ cells, 15% CD8CD3 cells and less than 0·5% CD4+ T cells (data not shown).

Cytokine mRNA measurement

RNA isolation, cDNA preparation and real-time PCR were performed as described previously [22]. Briefly, total RNA was isolated using the Ambion (Ambion, Austin, Texas) RNAqueous kit (PBMC). All samples were treated with DNase (Roche, Indianapolis, IN, USA) for 1 h at 37°C. cDNA was prepared using random hexamer primers (Amersham-Pharmacia Biotech Inc., Piscataway, NJ, USA) and M-MLV-reverse transcriptase (Invitrogen, Grand Island, NY, USA). Oligonucleotide primer and probe sequences for IFN-γ, IL-12, IL-2, IL-4, IL-6, TNF-α, MIP-1α and -β, and MDC were designed specifically for the TaqMan assay. All probes (Applied Biosystems) were 3′-labelled with TAMRA (6-carboxytetramethylrhodamine) and 5′-labelled with FAM (6-carboxyfluorescein), except the GAPDH (glyceraldehyde-3-phosphate-dehydrogenase) probe that was 5′ VIC-labelled. Primers and probe were used at a final concentration of 300 nM and 200 nM, respectively. Samples were tested in duplicate and the PCR for the housekeeping gene GAPDH and the target (cytokine) gene were run in parallel on the same plate. The cytokines TNF-α, IFN-γ, and the chemokines MIP-1α and -β, and MDC were run in multiplex assays in which the cytokine gene and the GAPDH sequence were amplified in the same tube. Primer concentrations were adjusted to 60 nM for GAPDH and to 900 nM for the cytokine gene. The reaction was carried out on a 96-well optical plate (Applied Biosystems, Foster City, CA, USA) in a 25-μl reaction volume containing 5 μl cDNA + 20 μl Mastermix (Applied Biosystems). All sequences were amplified using the 7700 default amplification program: 2 min at 50°C, 10 min at 95°C, followed by 40–45 cycles of 15 s at 95°C and 1 min at 60°C. Results were analysed with the SDS 7700 system software, version 1·6.3 (Applied Biosystems) on a G4 Macintosh computer (Apple Computer, Cupertino, CA, USA). Cytokine mRNA expression levels were calculated from delta cycle threshold (ΔCt) values, and are reported as fold increase (FI) of cytokine mRNA levels in hormone-treated cells compared to untreated cells. Ct values correspond to the cycle number at which the fluorescence due to enrichment of the PCR product reaches significant levels above the background fluorescence (threshold). In this analysis, the Ct value for the housekeeping gene (GAPDH) is subtracted from the Ct value of the target (cytokine) gene. The delta Ct (ΔCt) value for the hormone-treated sample is then subtracted from the delta Ct value of the corresponding untreated sample (ΔΔCt). Assuming that the target gene (cytokine) and the reference gene (GAPDH) are amplified with the same efficiency (data not shown), the FI in hormone-treated samples compared to untreated samples is then calculated as: FI = 2ΔΔCt (User Bulletin no. 2, ABI Prism 7700 Sequence Detection System (Applied Biosystems)).

Statistical analysis

For analysis of the ELISPOT data, the animals were divided into three groups based on the stage of the menstrual cycle at the time of necropsy (see above). Results are reported as the mean of the group (±standard error, s.e.). Single factor analysis of variance (ANOVA) was used to determine if the variation among the means of the three groups for each tissue was statistically significant. If ANOVA analysis indicated a significant difference (P < 0·05), then the Tukey–Kramer post-hoc test was used in a pairwise comparison between groups to determine if the difference between the means of two groups for each tissue was statistically significant at the 95% confidence level.

RESULTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Frequency of ISC in cervical and vaginal mucosa at different stages of the menstrual cycle

The frequency of IgG-ISC was markedly higher than the frequency of IgA-ISC in cervix and vagina (Figs 1 and 2). In the cervix, the frequency of IgG-ISC in Group II was significantly higher than the frequency of IgG-ISC in Group I or Group III (Fig. 1; P-values are noted in the figure). In the vagina, although the difference in the frequency of IgG-ISC among the three animal groups was not significant, a trend toward a higher frequency of IgG-ISC in Group II was clear (Fig. 1). In the cervix and vagina, the frequency of IgA-ISC in Group II animals was significantly higher than the frequency of IgA-ISC in Group I or Group III (Fig. 2; P-values are noted in the figure). Thus, the frequencies of both IgG and IgA-ISC in the lower genital tract change during the menstrual cycle with the highest frequency of ISC found occurring during the periovulatory period.

image

Figure 1. The frequency of IgG-ISC in tissues collected at three stages of the menstrual cycle. Each bar indicates the mean number of spontaneous IgG-ISC per 106 mononuclear cells in a tissue of animals assigned to one of three groups. Standard error bars are shown. The numbers in the row at the top of the figure indicate the P-value generated using ANOVA analysis to compare the means of the three animal groups. A P-value of less than 0·05 (denoted by *) indicates that the differences in the mean number of ISC among the three groups is significant in that tissue. Significant differences (P < 0·05) found by pairwise comparison between two groups, as determined by the Tukey–Kramer test, are noted by ** on the dendrogram above the bars of the two groups being compared. n.s. indicates no significance by the Tukey–Kramer test. Cx, cervix; VAG, vagina; Iliac, iliac lymph node; SPL, spleen; ING, inguinal lymph node; Ax, axillary lymph node; MES, mesenteric lymph node; PBMC, peripheral blood mononuclear cells; BM, bone marrow; TON, tonsil. Animal groups: bsl00022, Group I (n = 7); bsl00036, Group II (n = 4); ▪, Group III (n = 7).

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image

Figure 2. The frequency of IgA-ISC in tissues collected at 3 stages of the menstrual cycle. Each bar indicates the mean number of spontaneous IgA-ISC per 106 mononuclear cells in a tissue of animals that were assigned to one of three groups. Standard error bars are shown. The layout of the figure is described in the Fig. 1 legend. (*) and (**) denote statistically significant differences (see Fig. 1 legend for explanation of layout). Animal groups: bsl00022, Group I (n = 7); bsl00036, Group II (n = 4); ▪, Group III (n = 7).

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Frequency of ISC in lymph nodes (LNs), PBMC, spleen, tonsil and bone marrow (BM) at different stages of the menstrual cycle

There were higher frequencies of IgG-ISC than IgA-ISC in all lymphoid tissues tested. The frequency of IgG-ISC in all tissues varied with the stage of the menstrual cycle (Fig. 1). The spleen, axillary LN, mesenteric LN and PBMC of Group II animals had a significantly higher frequency of IgG-ISC compared to the same tissues from animals in Groups I and III (P < 0·05, Tukey–Kramer). Although the differences did not reach the level of statistical significance, in all other anatomic sites except the inguinal LN, the highest frequency of IgG-ISC was found consistently in the Group II animals (Fig. 1).

IgA-ISC were found in all the tissues examined (Fig. 2). The spleen, inguinal LN, axillary LN, PBMC and tonsil of Group II animals had a significantly higher frequency of IgA-ISC than the same tissues of the animals in Groups I and III. Although the differences in the frequency of IgA-ISC in iliac LN and mesenteric LN did not reach the level of statistical significance, the frequency of IgA-ISC in these anatomical sites was also the highest in Group II animals (Fig. 2). This trend was not seen in the bone marrow.

Frequency of anti-TT, KLH and CT AbSC in cervical and vaginal mucosa at different stages of the menstrual cycle

Intramuscular immunization with TT induced a high frequency of anti-TT IgG-AbSC in the cervical and vaginal mucosa. The frequency of anti-TT IgG-AbSC in the lower genital tract ranged from 1000 to 4000per 106 mononuclear cells. Although the difference in the frequency of anti-TT IgG-AbSC in the cervix and vagina among the three animal groups was not significant, a trend toward a higher frequency of anti-TT IgG-AbSC in Group II compared to the other two animal groups was apparent (Fig. 3). Intramuscular immunization with TT or KLH and oral immunization with CT failed to induce detectable antigen specific IgA-AbSC in the cervical or vaginal mucosa.

image

Figure 3. The frequency of anti-TT IgG-AbSC in tissues collected at 3 stages of the menstrual cycle. Each bar indicates the mean number of spontaneous anti-TT IgG-AbSC per 106 mononuclear cells in each tissue from animals assigned to one of three groups. Standard error bars are shown. The layout of the figure is described in the Fig. 1 legend. (*) and (**) denote statistically significant differences (see Fig. 1 legend for explanation). Animal groups: bsl00023, Group I (n = 7); bsl00036, Group II (n = 4); ▪, Group III (n = 7).

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Frequency of anti-TT AbSC in LNs, PBMC, spleen, tonsil and BM at different stages of the menstrual cycle

Anti-TT IgG-AbSC were detected in all the tissues studied (Fig. 3). In most tissues anti-TT AbSC were highest for Group II animals. Statistically significant differences in AbSC frequency among the three groups were observed in the spleen, inguinal LN, axillary LN and BM (Fig. 3). There were no statistically significant differences in AbSC frequency in cervix, vagina, mesenteric LN or tonsil, although group II monkeys showed a trend toward a higher frequency of anti-TT IgG-AbSC in all these tissues (Fig. 3). The highest frequency of anti-TT IgG-AbSC was found in the iliac LN. The iliac LN receive afferent lymphatics originating in the upper leg, the site of the intramuscular TT immunization. In the iliac LN, there was not a significant difference in anti-TT IgG-AbSC frequency among the three groups, although Group II did have the highest frequency of anti-TT ABSC.

Anti-TT IgA-AbSC were present in iliac LN, inguinal LN, axillary LN, spleen and BM. The highest frequency of anti-TT IgA-AbSC was in the iliac LN. Group II animals had a significantly higher frequency of anti-TT IgA-AbSC than animals in Groups I or III in the iliac LN, axillary LN and spleen (P < 0·05) (data not shown). Although this trend was also found in the inguinal LN and BM, the differences observed were not statistically significant (data not shown).

Frequency of anti-KLH AbSC in LN, PBMC, spleen and BM at different stages of the menstrual cycle

Anti-KLH IgG-AbSC were found mainly in iliac LN (80–850/106 MNC) and spleen (24–95/106 MNC). Only a few cells (2–6/106 MNC) were detected in the inguinal LN, axillary LN, PBMC and BM. There was no significant difference in anti-KLH IgG-AbSC frequency in iliac LN and spleen of the three groups. However, a trend toward a higher frequency of KLH IgG-AbSC in Group II animals was apparent (data not shown). Although anti-KLH IgA-AbSC were detected in iliac LN (10–168/106 MNC), inguinal LN (2–4/106 MNC), a×illary LN (2–6/106 MNC) and spleen (2–4/106 MNC), a significant difference in anti-KLH IgA-AbSC frequency among the three animal groups was not seen. However, a trend toward a higher frequency of KLH IgA-AbSC in both these tissues of the group II animals was apparent. The iliac lymph node contained higher numbers of anti-KLH IgA-AbSC than other tissues (data not shown). The localization of TT and KLH AbSC in the iliac LN is consistent with the upper leg being the site of all intramuscular immunizations. Based on these results, immunization with recall antigens in the upper leg results in antigen-specific B-cell proliferation and differentiation of AbSC in the iliac LN.

Frequency of anti-CT AbSC in mesenteric LN at different stages of the menstrual cycle

After oral immunization with CT, anti-CT IgG-AbSC were found only in the mesenteric LN (Fig. 4). The frequency of anti-CT AbSC was low (5–15/106 MNC) compared to the response elicited by immunization with the other two antigens. A low frequency of anti-CT IgA-AbSC (2–5/106 MNC) was detected only in the mesenteric LN (Fig. 4). No difference between the frequency of CT-specific AbSC was detected among the three animal groups. However, the very low frequency of CT-specific AbSC in the mesenteric LN, at all stages of cycle, made it difficult to detect any differences in AbSC frequency.

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Figure 4. The frequency of anti-CT AbSC in mesenteric LN collected at 3 stages of the menstrual cycle. (a) Anti-CT IgG-AbSC. (b) Anti-CT IgA-AbSC. Each bar indicates the mean number of spontaneous anti-CT AbSC per 106 mononuclear cells from animals assigned to one of three groups. Standard error bars are shown. The mean number of anti-CT IgG- and IgA-AbSC in the three groups was not significantly different by ANOVA analysis. Animal groups: bsl00026, Group I (n = 7); bsl00036, Group II (n = 4); ▪, Group III (n = 7).

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Dose-dependent effect of progesterone and oestradiol on the frequency of ISC in vitro

The higher frequency of ISC in the lower genital tract and other tissues during the periovulatory stage compared to the luteal and menstrual stages could be explained simply if changes in oestrogen and progesterone levels were responsible for the variation in B cell activity during the menstrual cycle. To test this hypothesis we exposed male macaque PBMC, highly enriched B cells and CD8+ T cell depleted-PBMC to either oestrogen or progesterone. Using PBMC from male animals minimized the effect of endogenous ovarian steroids on the cells. If PBMC from female animals had been used, these cells would have been exposed and responding to the oestrogen and progesterone levels in the donor female animal at the time the sample was drawn. This would have confounded our ability to control hormone-mediated effects by in-vitro addition of these hormones. Addition of progesterone, in concentrations spanning the physiological range found in female rhesus monkeys (ng/ml), to PBMC ELISPOT cultures for 18 h decreased the frequency of IgG and IgA-ISC in a dose-dependent manner (Fig. 5). Increasing the duration of exposure to progesterone from 18h to either 42h or 66 h led to further decrease in frequency of the IgA-ISC but had no effect on IgG-ISC frequency (data not shown). Addition of physiological doses (pg/ml) of oestradiol to PBMC cultures increased the frequency of IgA-ISC in a dose-dependent manner, except at the highest dose of oestrogen (1000 pg/ml), which produced a lower ISC frequency than the intermediate dose (Fig. 5). Identical doses of oestradiol had the same effect on the frequency of IgG-ISC in PBMC, but to a lesser extent (Fig. 5).

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Figure 5. The in vitro effect of progesterone and oestrogen concentrations on the frequency ISC in PBMC. (a) The effect of progesterone and oestrogen on the frequency of IgA-ISC. (b) The effect of progesterone and oestrogen on the frequency of IgG-ISC. Data are presented as the mean plus standard error of duplicate wells. The dotted line represents the baseline frequency of ISC in control wells (no steroids) which was set at 0. The numbers on the y axis indicate the percentage change in ISC frequency from baseline. The results are representative of more than 10 independent e×periments. (a) □, IgA-ISC/Prog.; ○, IgA-ISC/Est. (b) ○, IgG-ISC/Prog.; ▵, IgG-ISC/Est.

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The effect of ovarian steroid hormones on the frequency of ISC is indirect

To understand the role of steroids on B cell function, ISC frequencies were examined in B cell-enriched PBMC cultures (data not shown). The inhibitory effect of progesterone and the stimulatory effect of oestrogen on the frequency of IgG-ISC and IgA-ISC were not apparent in the B cell-enriched cultures (data not shown).

To characterize further the non-B cell population responsible for the observed effect of hormones on ISC frequency, we added oestrogen and progesterone to CD8+ T cell depleted-PBMC cultures. As with the B cell-enriched PBMC cultures, the exogenous hormones had no effect on the ISC frequency in CD8+ T cell depleted-PBMC cultures (data not shown). Although these results suggest strongly that CD8+ T cells are required for ovarian hormones to exert their effect on ISC frequency in these PBMC cultures, it should be noted that the anti-CD8+ magnetic beads that were used to deplete the CD8+ T cells also removed CD8+ NK cells. Thus it is possible that NK cells contributed to the observed effects.

Cytokine-expression patterns in hormone-treated CD8+ T cells

To define a potential role for cytokines in the hormonal regulation of immunoglobulin secretion by B cells, the cytokine mRNA profile for the proinflammatory (TNF-α, MIP-1α, MIP-1β, MDC) and the T helper (Th)1 (IL-12, IL-2, IFN-γ) and Th2 (IL-6, IL-4) cytokines was analysed in hormone-treated and untreated PBMC, CD8+-depleted cell fraction and CD8+-enriched cell fraction from PBMC cultures of two male rhesus macaques. In all cell populations, TNF-α, IL-2, IL-4, IL-6 and chemokine MIP-1α and -β mRNA expression levels were unchanged by the hormone treatments. The analysis of unfractionated PBMC and CD8-depleted PBMC revealed no differences in the cytokine mRNA expression profile between hormone-treated or untreated cultures. However, significant differences were observed between oestrogen-treated CD8+ positive cells and untreated CD8+ positive cells. Oestrogen treatment resulted in marked induction of IL-12 (6–8-fold increase over autologous hormone-naïve cells), MDC (13–34-fold increase over autologous hormone-naïve cells) and IFN-γ (12– 19-fold increase over autologous hormone-naïve cells) mRNA expression the CD8+ enriched cell fraction. In contrast, progesterone had no effect on the mRNA levels of the cytokines tested. To avoid artefactual alterations in gene expression caused by binding of monoclonal antibodies to cell surface receptors, we chose to use negative selection to enrich CD8+ T cells. However, this approach produced cell populations that contained approximately 80% CD8+ T cells and 15% CD8CD3 cells. Although the increase in IFN-γ mRNA expression is consistent with activation of a CD8+ T cell population, IL-12 expression occurs primarily in dendritic cells and MDC expression occurs in both DC and macrophages. Thus, the increased expression of IL-12 in the CD8-enriched fraction of oestrogen-pulsed PBMC cultures suggests that a DC population in the CD8+ enriched cell population responds to oestrogen exposure with increased expression of IL-12. The contribution made by these cytokines to increasing ISC frequency needs to be tested directly in in-vitro systems and it is probable that the hormone-regulated expression of genes in all the cell fractions is more complex than we were able to appreciate in our analysis. However, taken together, these data suggest that cytokines secreted by CD8+ T cells, and perhaps antigen presenting cells, play a role in the observed effects of hormones on the frequency of ISC.

DISCUSSION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

In the present study we demonstrate, for the first time, that the frequency of ISC and AbSC in the genital tract mucosa and systemic lymphoid tissues vary markedly at different stages of the menstrual cycle of primates. The frequency of IgG and IgA-ISC (specific and total) in the cervico-vaginal tissues was highest during the periovulatory stage and lowest during the luteal phase and menstruation. We also provide the first evidence that these changes in the frequency of ISC occur in lymphoid tissues throughout the body during the menstrual cycle. Further, the in-vivo results are consistent with the findings of in vitro experiments. In vitro, oestrogen consistently increased the frequency of ISC (both IgG and IgA), while progesterone decreased the frequency of ISC in rhesus monkey PBMC. The effect of the ovarian steroids on monkey B cells was dose-dependent and required the presence of CD8+ T cells in the cultures. This result is consistent with the published findings that oestrogens enhance non-specific differentiation of human antibody-secreting cells in vitro[14,15] by inhibition of suppressor T cells [14]. As we cannot exclude the possibility that CD8+ NK cells contribute to the observed effects, experiments are under way to define further the CD8+ cell subset that is responsible for altering ISC frequency. However, in vitro studies in rhesus monkeys and humans support the hypothesis that CD 8+ T cells can regulate Ig secretion by B cells in response to changes in ovarian hormone levels. These observations are also the first direct experimental evidence for an in vivo role of ovarian sex steroids and CD8+ T cells on ISC frequency in lymphoid tissues of menstrual monkeys and, by extension, women.

While our in vivo findings are in agreement with the in vitro studies, the timing of peak ISC frequency at ovulation was surprising. Previous reports, including our own, have shown that the levels of IgG and IgA in cervico-vaginal secretions of humans and rhesus macaques are the lowest around ovulation [19,23,24]. There may be several explanations for the discordance between the relatively low Ig levels in cervico-vaginal secretions and the high frequency of ISC in the lower genital tract mucosa at mid-cycle. The most likely explanation is that the increase in local Ig production does not compensate for the dilution effect caused by the increase in the production of cervical mucus at mid-cycle [24–26]. In addition, the stratified squamous epithelium of the vagina and ectocervix of these animals, which is the primary barrier to diffusion of IgG and monomeric IgA from the lamina propria to vaginal secretions, is thickest at ovulation [20]. Although the concentrations of IgG and IgA in serum are not influenced by the menstrual cycle [19], the frequency of ISC in numerous tissues was significantly higher at mid-cycle than at other stages of the cycle. Similar trends toward a higher frequency of ISC at mid-cycle, although not statistically significant, were seen in most other tissues examined. Reconciling the stable serum Ig levels with the observed changes in ISC frequency is straightforward given that the half-life of IgG and monomeric IgA are 22 and 6 days, respectively [27]. The relatively long half-life of serum Ig would mask changes in Ig levels that might be caused by the cyclic variations in number of ISC.

The observed changes in ISC frequency could reflect changes in the relative frequency of other mononuclear cell populations in the tissues. However, quantitative morphometric analysis of mononuclear cell populations demonstrated that there were no significant differences in the size or composition of the lymphocyte, macrophage or dendritic cell populations within the lower genital tract tissues of the 18 animals in this study [20]. In addition, there are no significant changes in the absolute number of lymphocytes or the relative frequencies of lymphocyte subsets in the peripheral blood of female rhesus macaques that could explain the observed shifts in ISC frequency during the menstrual cycle (Miller, unpublished data).

In cultures of rodent and human PBMC, oestrogen stimulates pokeweed mitogen-induced B cell differentiation [14,28]. The immunostimulatory effect was thought to have been mediated by the inhibitory effect of oestrogen on a T suppressor cell population [29]. The results of the in-vitro experiments with monkey PBMC suggest that the immunostimulatory effect of oestrogen on B cells is CD8+ T cell-mediated. We also found that oestrogen treatment resulted in an up-regulation of IL-12, IFN-γ and MDC mRNA in CD8 positive cells. The up-regulation of IFN-γ mRNA suggests a CD8+ T cell-dependent, cytokine-mediated mechanism of oestrogen action. Consistent with this, it has been shown in that human CD8+ T cells, but not CD4+ T cells, express oestrogen receptors [30]. In addition, in mice oestrogen directly increases the activity of the IFN-γ promoter in lymphoid cells [31]. Our results are also in agreement with data reporting an increased secretion of IFN-γ in stimulated and unstimulated human PBMC after oestrogen treatment [32].

IL-12 is a cytokine produced by macrophages and dendritic cells (DC) [33–35]. There are no reports of IL-12 production by CD8+ T cells, thus it difficult to explain the increased IL-12 expression found in the oestrogen-stimulated CD8+-enriched cultures. The simplest explanation is that antigen-presenting cells (APCs) were present in the CD3CD8 cells that comprised 15% of the CD8+ cell-enriched fraction or that CD8αα+ DC were present in the CD8+-enriched cultures. Indeed, based on these considerations, it seems very likely that oestrogen stimulates IL-12 secretion in antigen-presenting, possibly dendritic cells (DC). It has been shown that DC-derived IL-12 is necessary for the differentiation of activated naïve B cells into plasma cells [36] and it is known that macrophages express oestrogen receptors [37]. Thus, the source of the MDC and IL-12 in the CD8+ enriched fraction of oestrogen-stimulated rhesus monkey PBMC cultures could include APCs. It remains to be determined if oestrogen effects the number of immunoglobulin-secreting B cells via cytokine secretion by regulatory CD8 positive T cells alone or if DC are also involved.

Although we did not determine the mechanisms by which hormones regulate ISC frequency, a plausible explanation is that oestrogen increases the frequency of ISC by stimulating regulatory CD8+ T cells to increase expression of IFN-γ as observed. Increased IFN-γ expression in turn results in the improved differentiation of B cells and/or increased production of Ig by differentiated B cells. APCs secreting IL-12 may contribute to this T cell/B cell interaction. By contrast, we did not detect altered cytokine expression in progesterone-stimulated CD8+ T cells, suggesting that these soluble mediators do not mediate the progesterone-mediated decrease in ISC frequency. In mice, there are several models of Ig suppression due to MHC I-restricted CD8+ T cell-mediated lysis of B cells [38–42]. By analogy, the progesterone-mediated decline in ISC cell frequency may be due to lysis of B cells by CD8+ T cells. Experiments to distinguish among these possibilities are under way.

The study also produced significant insight into the anatomical location of anamnestic B cell responses. The frequency of ISC found in the PBMC of the animals in this study is within the range described previously for humans and macaques. A comparison of the frequency of IgG-ISC in human and monkey PBMC as detected by ELISPOT assays developed in various laboratories, including ours, is provided in Table 2. Healthy humans [43] and rhesus macaques have a similar number of Ig-ISC in PBMC (Table 2). We did, however, find a very high number of anti-TT AbSC in the iliac lymph nodes of experimental animals (Fig. 3). This finding probably reflects the fact that TT is a recall antigen for all monkeys at the CRPRC, as they are immunized routinely with TT beginning at a young age. Thus, it is not surprising that the frequency of AbSC specific to the recall antibody, TT, was higher in the tissues tested than the frequency of AbSC producing antibodies against KLH, a novel Ag. The high frequency of anti-TT AbSC and anti-KLH AbSC in the iliac lymph nodes is consistent with the observation that multiple intramuscular immunizations induce the strongest immune response in local draining lymph nodes compared to other tissues [44].

Table 2.  IgG-immunoglobulin secreting cell frequency in human and non-human primate PBMC
SpeciesImmune statusnIgG-ISC/106 MNCBoost to sampling intervalReferences
HumanNormal6250 (47)
HumanNormal18100 (48)
HumanNormal3260 ± 74 (49)
HumanAcute HIV infection41500<90d(47)
HumanChronic HIV infection1125004 months–12 years(47)
Cynomologous monkeysNormal2255 ± 115 (43)
Cynomologous monkeysAnamnestic (3×)55850 ± 26507d(43)
Rhesus monkeysNormal2079 ± 18 Miller, Lü (unpublished)
Rhesus monkeysImmunized (3×)5344 ± 55>21dMiller, Lü (unpublished)
Rhesus monkeysAnamnestic178541 ± 28717dSee results

The frequency of anti-TT IgG-AbSC in the cervico-vaginal mucosa and iliac lymph node did not vary significantly during the menstrual cycle, although the frequency of anti-TT IgG-AbSC in the cervico-vaginal mucosa was higher in Group II than in other groups. Hormone-induced variation in the anti-TT IgG-AbSC frequency may have been masked by the proliferation of anti-TT B cells during the strong and ongoing anamnestic response to the booster TT immunization. While anti-TT IgA-AbSC were undetectable in the cervico-vaginal mucosa, they were found in the iliac lymph nodes and the frequency of anti-TT IgA-AbSC was significantly higher in iliac lymph nodes of Group II animals compared to the other two groups.

In a separate and unrelated study, we previously reported [45] that oral CT immunization induced anti-CT AbSC formation predominantly in the mesenteric lymph nodes, but failed to induce ISC in the lower reproductive tract. The results of the current study further support that result, as anti-CT AbSC were found only in the mesenteric lymph nodes. It is important to note that in the current study, intramuscular (upper leg) immunization induced a high frequency of anti-TT IgG AbSC in the cervico-vaginal mucosa. We have also found that intranasal immunization of rhesus macaques induces antigen-specific B cell immunity in the lower genital tract [46]. Thus it appears that immunization in some, but not all, mucosal or intramuscular sites can result in the localization of antigen-specific B cells in the cervico-vaginal mucosa of primates.

Although there have been numerous published in-vitro studies demonstrating that sex steroid hormones can effect human B-cell differentiation and function, there have been no reports establishing an in-vivo role for sex steroids in regulating B cell immunity of menstrual primates. In the current study, we show that, in rhesus monkeys, the frequency of ISC and AbSC in the genital tract tissues and numerous systemic lymphoid tissues are affected significantly by the stage of the menstrual cycle. Further, we found that the observed effect of oestrogen and progesterone on B cell physiology in-vitro is mediated indirectly through CD8+ T cells. Studies are under way to determine if a similar immune regulatory pathway exists in women. Autoimmune diseases are more common in women and many of these diseases are associated with a polyclonal activation of B cells or the production of self-reactive antibodies. In addition, it has proven to be very difficult to elicit protective immunity against sexually transmitted diseases in women. A better understanding of the hormone-mediated molecular and cellular regulatory pathways that control antibody secretion by B cells in females may lead to therapeutic interventions for immune-mediated diseases and vaccine strategies that are capable of protecting women, and their infants, from STDs.

ACKNOWLEDGEMENTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

We thank Michael Torten for editorial assistance, Kristen Bost, Steve Joye, Lisa Laughlin, Linda Hirst and David Bennett for technical assistance and Nicholas Lerche for advice on statistical analysis. This work was supported by National Institutes of Health grants: NICHD RO133169; NCRR P51 AG00169; NICHD U54 29125 and NCRR RR14555.

REFERENCES

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  2. SUMMARY
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
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